Development and applicability of a ready-to-use PCR system for GMO screening

Rosa, Sabrina; Gatto, Francesco; Angers-Loustau, Alexandre; Petrillo, Mauro; Joachim, Kreysa; Querci, Maddalena

doi:10.1016/j.foodchem.2016.01.007

Cited by 40 publications

(28 citation statements)

References 24 publications

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“…In terms of qualitative PCR assays, prespotted plates have been effectively used for detection of multiple GMOs in various crop plants [16, 17]. These are plastic plates used in qualitative PCR with primers and probes for selected assays predispensed in wells.…”

Section: Overview Of Different Technologies Used For Gmo Detectionmentioning

confidence: 99%

“…These are plastic plates used in qualitative PCR with primers and probes for selected assays predispensed in wells. Hands-on time is shortened because only one reaction mixture needs to be prepared per sample, which is then distributed over several wells [16]. Element-specific, taxon-specific and event-specific prespotted plates can be prepared and used for the detection of multiple GMOs.…”

Section: Overview Of Different Technologies Used For Gmo Detectionmentioning

confidence: 99%

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Critical assessment of digital PCR for the detection and quantification of genetically modified organisms

Demeke¹,

Dobnik

2018

Anal Bioanal Chem

110

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The number of genetically modified organisms (GMOs) on the market is steadily increasing. Because of regulation of cultivation and trade of GMOs in several countries, there is pressure for their accurate detection and quantification. Today, DNA-based approaches are more popular for this purpose than protein-based methods, and real-time quantitative PCR (qPCR) is still the gold standard in GMO analytics. However, digital PCR (dPCR) offers several advantages over qPCR, making this new technique appealing also for GMO analysis. This critical review focuses on the use of dPCR for the purpose of GMO quantification and addresses parameters which are important for achieving accurate and reliable results, such as the quality and purity of DNA and reaction optimization. Three critical factors are explored and discussed in more depth: correct classification of partitions as positive, correctly determined partition volume, and dilution factor. This review could serve as a guide for all laboratories implementing dPCR. Most of the parameters discussed are applicable to fields other than purely GMO testing. Graphical abstractThere are generally three different options for absolute quantification of genetically modified organisms (GMOs) using digital PCR: droplet- or chamber-based and droplets in chambers. All have in common the distribution of reaction mixture into several partitions, which are all subjected to PCR and scored at the end-point as positive or negative. Based on these results GMO content can be calculated.

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Section: Overview Of Different Technologies Used For Gmo Detectionmentioning

confidence: 99%

Section: Overview Of Different Technologies Used For Gmo Detectionmentioning

confidence: 99%

Critical assessment of digital PCR for the detection and quantification of genetically modified organisms

Demeke¹,

Dobnik

2018

Anal Bioanal Chem

110

View full text Add to dashboard Cite

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“…While the cost is influenced by several factors, such as sample type, composition and analytical technology used (Milavec et al 2014), decreasing the number of full quantitative assays has a direct effect on the overall cost. This can be illustrated using the data generated in a recent study (Rosa et al 2016), for which the composition of 135 real-life samples containing GM events was communicated to our laboratory. Results of quantifications performed by laboratories showed that ∼40 % of the GM events were at 0.1 % or not quantifiable (i.e.…”

Section: Practicability and Impactmentioning

confidence: 99%

“…To demonstrate the feasibility of the approach, six maize GM events were chosen based on their frequency of occurrence in EU official samples (Rosa et al 2016). The associated certified reference materials (CRMs) were tested at different GM concentration levels by RTi-PCR for the taxon-and the event-specific assays and Cq signals were then combined to calculate ΔCq values (Cq GM -Cq REF ).…”

Section: Introductionmentioning

confidence: 99%

Semi-Quantification of GM Maize Using Ready-To-Use RTi-PCR Plates

et al. 2016

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European Union (EU) legislation foresees that food and feed are labelled for their genetically modified organism (GMO) content when the ingredients contain authorised GMOs in a proportion higher than 0.9 %. Non-authorised GMOs are not allowed on the EU market. Exception is made for feed materials containing traces of GM events lawfully placed on the market in non-EU countries and for which an authorisation is pending in the EU or expired: in these cases, a minimum required performance limit (MRPL) of 0.1 % is applied. Considering that the number of GMOs placed on the EU market is constantly growing, laboratories need to expand their capacity accordingly to be able to identify and, if necessary, quantify any GM material. To this purpose, our laboratory had previously developed a multi-target ready-touse system, also known as a pre-spotted plate (PSP), which allows the qualitative detection of up to 44 GM events in one single real-time (RTi) PCR experiment. Should any event be detected, the laboratory would proceed with its quantification. This study evaluates the possibility to use the data generated by the PSP system in a semi-quantitative manner, allowing a categorisation of the GM quantity in the sample. ΔCq values were calculated and modelled via linear regression to estimate limits indicating whether the GM content is (1) above, (2) below or (3) near a defined quantity and thus requires further quantification. Identifying the GM events present in a sample and simultaneously discriminating whether it needs or not further quantification would offer to testing laboratories a valuable gain in time and resources. Six maize GM events frequently found in food and feed were selected for this evaluation (Bt11, MON810, MON88017, MON89034, NK603 and TC1507). The feasibility of the approach was successfully verified in-house using spiked and proficiency test samples.

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“…Also, pre-spotted screening and event plates have been developed [34,36] to facilitate the screening and identification of GMOs in food and feed products.…”

mentioning

confidence: 99%